Abstract

Repeatedsocialdefeat (RSD) is a murine stressor that recapitulates key physiological, immunological, and behavioral alterations observed in humans exposed to chronic psychosocial stress. Psychosocial stress promotes prolonged behavioral adaptations that are associated with neuroinflammatory signaling and impaired neuroplasticity. Here, we show that RSD promoted hippocampal neuroinflammatory activation that was characterized by proinflammatory gene expression and by microglia activation and monocyte trafficking that was particularly pronounced within the caudal extent of the hippocampus. Because the hippocampus is a key area involved in neuroplasticity, behavior, and cognition, we hypothesize that stress-induced neuroinflammation impairs hippocampal neurogenesis and promotes cognitive and affective behavioral deficits. We show here that RSD caused transient impairments in spatial memory recall that resolved within 28 d. In assessment of neurogenesis, the number of proliferating neural progenitor cells (NPCs) and the number of young, developing neurons were not affected initially after RSD. Nonetheless, the neuronal differentiation of NPCs that proliferated during RSD was significantly impaired when examined 10 and 28 d later. In addition, social avoidance, a measure of depressive-like behavior associated with caudal hippocampal circuitry, persisted 28 d after RSD. Treatment with minocycline during RSD prevented both microglia activation and monocyte recruitment. Inhibition of this neuroinflammatory activation in turn prevented impairments in spatial memoryafter RSD but did not prevent deficits in neurogenesis nor did it prevent the persistence of social avoidance behavior. These findings show that neuroinflammatory activation after psychosocial stress impairs spatial memory performance independent of deficits in neurogenesis and social avoidance.

SIGNIFICANCE STATEMENT:

Repeated exposure to stress alters the homeostatic environment of the brain, giving rise to various cognitive and mood disorders that impair everyday functioning and overall quality of life. The brain, previously thought of as an immune-privileged organ, is now known to communicate extensively with the peripheral immune system. This brain-body communication plays a significant role in various stress-induced inflammatory conditions, also characterized by psychological impairments. Findings from this study implicate neuroimmune activation rather than impaired neurogenesis in stress-induced cognitive deficits. This idea opens up possibilities for novel immune interventions in the treatment of cognitive and mood disturbances, while also adding to the complexity surrounding the functional implications of adult neurogenesis.

Increased inflammation and presence of CD45+ myeloid cells in the hippocampus after RSD. Mice were subjected to six cycles of RSD or left undisturbed as CONs. A, The mRNA levels of several inflammatory mediators (IL-1β, TNF, IL-6, and arginase) and growth factors (BDNF, VEGF, NGF, and IGF-1) were determined in the hippocampus collected immediately after RSD (n = 6). In a related experiment, mice were subjected to six cycles of socialdefeat, and mice were perfused and fixed with 4% paraformaldehyde 14 h later. Brain samples were postfixed, frozen, and sectioned, and Iba-1 or CD45 expression was determined (n = 6). B, Representative images of Iba-1 labeling are shown in the DG. The arrows show the Iba-1+ cell depicted in the inset. C, Quantification of Iba-1 labeling for the entire volume of the hippocampus with rostral–caudal distinction. D, Representative images of CD45 labeling are shown in the DG. The arrows show the CD45+ cell depicted in the inset. E, Quantification of CD45+ cells per hippocampal section. Data represent mean ± SEM. *p < 0.05, means are significantly different from CON.

Impaired working memoryafter RSD. A, Representative time line of working memory assessment in the MWM. Mice were subjected to six cycles of RSD or left undisturbed as CONs. Working memory was examined for 5 d (D7–D11) after completion of RSD (n = 10). B, In this paradigm, the escape platform was moved to a different quadrant each testing day. C, Representative paths for CON and RSD mice in the MWM on day 3. Velocity (D), latency to the platform (E), distance traveled (F), and percentage time in the outer annulus (G) were determined for all 5 testing days. Graphs represent the mean ± SEM. *p < 0.05, means are significantly different from CONs; #p = 0.06–0.10, means tended to be different from CONs.

Impaired spatial memory recall in the MWM after RSD. A, Representative time line of learning and memory recall assessments in the MWM. Mice were subjected to six cycles of RSD or left undisturbed as CONs. Learning was examined after the completion of RSD for 5 d (D7–D11), and then memory recall was tested (D12; n = 8). B, In this paradigm, the escape platform remained in the same location each testing day of the acquisition phase but was removed during the probe trial. During the acquisition phase, latency to the platform (C) and distance traveled (D) were determined each of the 5 d of testing. E, Representative search paths of CON and RSD mice during the probe trial (D12). Spatial memory recall was assessed during the probe trial, and distance traveled (F) and percentage of time in the target quadrant (G) were determined. Graphs and bars represent the mean ± SEM. *p < 0.05, means are significantly different from CONs.

Impaired spatial memory recall in the Barnes maze after RSD. A, Representative time line of cognitive assessment in the Barnes maze after RSD. Mice were trained on the Barnes maze to acquire learning before socialdefeat (Pre-Con and Pre-RSD). Next, mice were subjected to six cycles of RSD or left undisturbed as CONs, and memory recall was determined on D7 and D8. Escape latency (B) and total errors (C) are shown before and after RSD exposure. In a related experiment, mice were used as above, and memory recall was determined at 34 and 35 d. Escape latency (D) and total errors (E) are shown before and after RSD exposure. Lines represent the mean ± SEM. *p < 0.05, means are significantly different from CONs; #p = 0.06–0.10, means tended to be different from CONs.

Number of proliferating progenitor cells and young neurons in the hippocampus remain unchanged 0.5 d after RSD. Mice were subjected to six cycles of RSD or left undisturbed as CONs. BrdU was injected during the last three cycles of socialdefeat. BrdU+ and DCX+ cells were determined in the DG 0.5 d later (n = 6). A, Representative images of BrdU labeling in the DG for CON and RSD mice. B, Quantification of BrdU labeling in the DG for the entire volume of the hippocampus. C, Representative images of DCX+ neurons in the DG for CON and RSD mice. D, Quantification of DCX labeling in the DG for the entire volume of the hippocampus. E, Representative images of mature and immature DCX+ neurons. F, Quantification of immature DCX labeling and mature DCX labeling. Bars represent the mean ± SEM.

Reduced proliferating neurons in the hippocampus 10 d after RSD. Mice were subjected to six cycles of RSD or left undisturbed as CONs. BrdU was injected during the last three cycles of socialdefeat. BrdU+ and DCX+ cells were determined in the DG 10 d later (n = 6). A, Representative images of BrdU/DCX double labeling in the DG are shown. The insets represent a BrdU+/DCX+ cell (yellow) and BrdU+/DCX− cell (green). B, Percentage of BrdU+ cells that were also DCX+ in the DG. Bars represent the mean ± SEM. *p < 0.05. means are significantly different from CON.

Impaired development of hippocampal NPCs into mature neurons 28 d after RSD. Mice were subjected to six cycles of RSD or left undisturbed as CONs. BrdU was injected during the last three cycles of socialdefeat. BrdU+, NeuN+, and GFAP+ cells were determined 28 d later (n = 6). A, Representative images of BrdU labeling are shown in the DG. B, Quantification of BrdU+ cells per section of the hippocampus. C, Representative images of NeuN/BrdU labeling are shown in the DG. The arrows show the cell depicted in the inset. D, Quantification of NeuN+/BrdU+ cells per section of the hippocampus with rostral–caudal distinction. E, Representative images of GFAP/BrdU labeling are shown in the DG. The arrows show the cell depicted in the inset. F, Quantification of GFAP+/BrdU+ cells per section of the hippocampus. Bars represent the mean ± SEM. *p < 0.05, means are significantly different from CON.

Social avoidance behavior was present 28 d after RSD. Mice were subjected to six cycles of RSD or left undisturbed as CONs, and social interaction was determined 28 d later (n = 14). A, Representative time line of the experiment is shown. B, Interaction paths are shown for the Empty (Trial 1, top) and Social (Trial 2, bottom) trials. C, Time spent in the interaction zone during the Empty and Social Trials. D, Time spent in the corner zone during the Empty and Social Trials. Bars represent the mean ± SEM. *p < 0.05, means are significantly different from all other groups.

Minocycline intervention attenuated RSD-induced microglia activation and monocyte recruitment. Mice were subjected to six cycles of RSD or left undisturbed as CONs. Minocycline (90 mg/kg) was administered in drinking water throughout the 6 d of RSD. A, Representative images of Iba-1 labeling in the DG are shown. B, Proportional area quantification of Iba-1. C, Representative images of CD45 labeling in the DG are shown. B, Quantification of CD45+ cells. E, A coronal brain section was collected 14 h after the last cycle of stress, and the mRNA level of IL-1β was determined. Bars represent mean ± SEM. *p < 0.05, means are significantly different from CON.

Minocycline prevented spatial memoryimpairmentsafter RSD. Mice were trained on the Barnes maze to acquire learning before socialdefeat (Pre-Veh CON, Pre-Veh RSD, and Pre-Mino RSD). The Pre-Mino RSD group received minocycline (90 mg/kg) in drinking water 1 d before RSD and continued throughout the RSD period. Mice were subjected to six cycles of socialdefeat (Veh RSD and Mino RSD) or left undisturbed as CONs (Veh CON), and memory recall was determined on D7 and D8. A, Representative time line of cognitive assessment in the Barnes maze after RSD (n = 6–9). Escape latency (B) and errors (C) are shown before and after RSD exposure. *p < 0.05, means are significantly different from CON mice; #p = 0.06, mean tended to be different from CONs.

Minocycline did not prevent persistent social avoidance or long-term deficits in NPC differentiation. Mice were subjected to six cycles of RSD or left undisturbed as CONs. Minocycline (90 mg/kg) was administered in drinking water throughout the 6 d of RSD. All mice received BrdU injections (50 mg/kg) during the last three cycles of RSD. Then, 28 d later, social behavior was determined (D34). A, Representative time line of experimental manipulations (n = 6–9). B, Time spent in the interaction zone during the Empty and Social Trials was examined. C, Representative images of NeuN and BrdU labeling in the DG. The arrows show the cell depicted in the inset. D, Quantification of NeuN+/BrdU+ cells in the DG. Data represent the mean ± SEM. *p < 0.05, means are significantly different from CON mice.